PROJECT SUMMARY/ABSTRACT Anemia affects approximately 1.6 billion people worldwide, imposing an enormous burden on medical resources. Of the inherited anemias, the hemoglobinopathies, particularly sickle cell disease (SCD) and - thalassemia, stand out due to their prevalence and severity. The ability to postnatally elevate fetal hemoglobin (HbF) levels in SCD and -thalassemia via co-inheritance of positive genetic modifiers of HbF production or hydroxyurea (HU) treatment can significantly alleviate disease severity. However, HU is not without side effects and may even be carcinogenic. Novel therapies aimed at elevating HbF expression in adults are therefore desperately needed. Three major loci, including BCL11A, are known to modify HbF expression in humans. Together, however, they account for only ~50% of the variation in HbF levels. Hence, significant gaps in knowledge remain regarding the genetic control of HbF production. We will take advantage of two powerful mouse resources to identify novel regulators of -like globin switching, the mouse mutant Nan (neonatal anemia) and the newly developed high resolution Diversity Outbred (DO) mapping resource. In Nan, a single amino acid change in the second zinc finger of the erythroid Krppel-like factor (EKLF) causes sequence selective disruption of binding to a subset of its target genes. The result is severe anemia accompanied by a striking failure of hemoglobin switching. Expression of embryonic h1 globin is upregulated 100-fold in adult Nan spleen vs. wild type via a BCL11A independent mechanism. In highly genetically diverse DO mice, expression of h1 globin in adults varies substantially from individual to individual. Thus, these two resources, Nan and DO mice, possess the prerequisites required to detect novel regulators of -like globin switching using powerful, unbiased genetic (QTL mapping) and genomic (ChIP-seq, RNA-seq) strategies. The aim of this proposal is to identify novel genes regulating -like globin switching. To accomplish this aim, we will: (a) map modifiers of h1 expression in Nan F2 intercrosses and in DO mice; (b) perform ChIP-seq in erythroid populations to compare DNA targets differentially bound by wild type (WT) EKLF and mutant (Nan) EKLF, and RNA-seq to identify gene expression differences; and (c) analyze and integrate all data to identify, prioritize, and initiate analysis of candidate genes. ! Identifying genetic loci regulating h1 expression, differences in Nan- vs. WT-EKLF DNA targets, and differences in gene expression in Nan vs. WT erythroid cells will converge to identify novel regulators of - like globin switching, thereby providing important new therapeutic targets for the hemoglobinopathies. !